CONSTITUTIVE MEMBRANE ASSOCIATION POTENTIATES ACTIVATION OF BRUTON TYROSINE KINASE

Mutations in the nonreceptor tyrosine kinase Btk result in the B cell, immunodeficiencies X-linked agammaglobulinemia (XLA) in humans and X- linked immunodeficiency (xid) in mice. Genetic and biochemical evidenc e implicates Btk as a key component of several B cell signaling pathwa ys. Activation of Btk by a point mutation (E41K) within the PH domain (Btk) results in fibroblast transformation and is correlated with inc reased membrane localization of Btk. When wild type Btk is activated b y coexpression with Lyn, the tyrosine phosphorylated pool of Btk is hi ghly enriched in the membrane fraction. To determine whether membrane association is sufficient to activate Btk, we targeted Btk to the plas ma membrane using a series of fusion proteins including GagBtk, CD16Bt k and CD4Btk. Constitutive membrane association greatly enhanced the a bility of Btk to transform Rat2 fibroblasts in the presence of high le vels of Src activity. All membrane targeted forms of Btk were highly t yrosine phosphorylated. Transformation required membrane localization, Btk kinase activity, transphosphorylation by Src family kinases, and an intact SH2 domain but not the PH or SH3 domains. These data suggest that membrane localization is a critical early step in Btk activation .